In vitro transformation of petunia cells by an improved method of co-cultivation with A. tumefaciens strains |
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Authors: | Robert T Fraley Robert B Horsch Antonius Matzke Mary-Dell Chilton William S Chilton Patricia R Sanders |
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Institution: | (1) Monsanto Corporate Research & Development Staff, Biological Sciences, 800 N. Lindbergh, 63167 St. Louis, MO, U.S.A.;(2) Department of Biology, Washington University, 63130 St. Louis, MO, U.S.A.;(3) Institut für Molecular Biologie, Ostesreichische Akademie der Wissenschaften, A-5020 Salzburg, Austria;(4) CIBA-Geigy Biotechnology Facility, P.O. Box 12257, 27709 Research Triangle Park, NC, U.S.A.;(5) Department of Botany, North Carolina State University, 27650 Raleigh, NC, U.S.A. |
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Abstract: | Summary A method (termed co-cultivation) for transforming plant cells in vitro with A. tumefaciens strains, which was originally developed by Marton et al. (1978) Nature 277: 129–131, has been modified by the incorporation of a novel feeder plate culture system and been extended to use with petunia protoplasts. Using efficient cell plating and selection conditions for phytohormone-independent growth, large numbers of independent transformed calli can be obtained efficiently ( 10-1) and in less than 3 weeks following protoplast isolation. Southern hybridization analysis has confirmed that the majority of the resulting in vitro transformants contain a single copy of full length T-DNA.The high efficiency of this procedure allows simple screening to identify plant cells transformed by Ti plasmids attenuated by deletion of internal T-DNA regions. Results are presented that demonstrate the co-cultivation method can be used in conjunction with short term assays for monitoring plant gene expression. |
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